Retinoic acid is necessary for growth and differentiation of the organism and exerts its molecular actions by binding to specific nuclear receptors. Genetic approaches to study the effects of receptor gene inactivation by gene knockout technology have demonstrated redundancy in this family, i.e. inactivation of a single family member, for example retinoic acid receptor-a, is insufficient to produce a phenotype, i.e. a visible effect on the epithelium. We reasoned that ligand-knockout technology would be more telling of the effects of retinoic acid and estrogen on epithelial differentiation of mouse uterine and cervical epithelia. To create the ligand-retinoid knockouts we used female SENCAR mice maintained on a purified retinoid-deficient diet containing either 0 or 3 ?g of retinoic acid (for control)per gram of diet. To produce ligand-steroid knockout mice, the animals were ovariectomized. We tested the hypothesis that in the double knockout mice, i.e. ligand-estrogen and ligand-retinoid depleted, the phenotype of the single ligand-retinoid knockout, squamous metaplasia, is altered and that this may affect animal survival. Squamous cells arising in the normally simple columnar epithelium of the endocervix and uterine cavity were monitored by keratin K5 expression by immunohistochemistry. We found that ovariectomy did not change the time to onset of the phenotype squamous metaplasia caused by retinoid depletion. Instead, it prolonged survival of the ligand-retinoid knockout mice, by as much as 40%. This shows that factors other than epithelial differentiation per se control survival outcome of the ligand-retinoid knockout mice. In related studies, our previous work had shown that retinoic acid inhibits cell growth and induces apoptosis in estrogen receptor- positive MCF-7 and T-47D cells, but not in estrogen receptor-negative human breast carcinoma cell lines MB-231 and MD-453. We reasoned that these differences might be due to different uptake and metabolism of the drug by ER-positive compared to ER-negative cells. The two ER- positive cell lines reached maximum RA uptake at about 2 hr, followed by a sharp decline, so that most RA had disappeared from the cells and from the medium by 24 hr and was found as oxidation products in the culture medium. In contrast, the two ER-negative cell lines showed a pattern of lower accumulation without the sharp increase and subsequent steep decline, so that by 24 hr there was more RA in these cells and their culture medium than in the RA- responsive ER-positive cells, even though at 2 hr the ER-negative cells had taken up less RA than the ER- positive cells. Kinetic analysis of the uptake of RA in MCF-7 cells was consistent with rapid movement across the cell membranes and the actual rate determined by diffusion of albumin- bound retinoid to the cells. These studies are the first to demonstrate profound differences in retinoic acid accumulation and confirm previous results on different rates of RA metabolism between ER-positive and ER-negative human breast cancer cells. They, therefore, may introduce additional elements to be considered in the design of new drugs for cancer chemoprevention and therapy. - Carcinogenesis, Differentiation, Estrogens, Retinoids, Breast cancer ,